Single channel SCAM identifies pore -lining domains in connexin hemichannels

Abstract

Gap junctions are aggregates of intercellular channels which provide a direct pathway for electrical and metabolic signaling between coupled cells. To date, 21 connexin genes have been identified in humans, with tissue- and cell-specific but overlapping patterns of expression. Gap junction channels, composed of different connexin isoforms, display significant differences in conductance and charge selectivity among biological signaling molecules. Understanding the molecular basis for differences in connexin physiology requires the identification of the channel pore. We have applied the Substituted Cysteine Accessibility Method (SCAM) at the single channel level to identify pore lining residues and molecular determinants of open channel properties. The Cx46 hemichannel preparation allows us to use conventional ion channel biophysical techniques to assign pore lining residues based on their accessibility to sulfliydryl modification by methanthiosulfonate reagents in the open state in excised inside-out and outside-out patch configurations.;In Chapter 2, we demonstrate that residues D51, G46 and E43 at the amino end of the first extracellular loop (E1) are pore lining by their accessibility to positively charged, MTSET+ and MTSEA +, and negatively charged, MTSES-, reagents. The data show the electrostatic influence of oppositely charges reagents on ion flux. All three reagents modified all six connexin subunits. Accessibility continued into the extracellular end of TM1 at A39 and L35. Residues L35 through G46 fall on one face of an a-helix while D51 is likely in a transitional zone in the extracellular gap. Chapter 3 describes the sensitivity of the cytoplasmic half of TM1 (F32-V24) to cysteine substitution which have so far precluded its assignment to the pore. We also demonstrate that while all six connexin subunits can be modified by small MTS reagents, modification is limited to fewer subunits with larger, uncharged MTSEA-BIOTIN reagents. In Chapter 4 we describe studies of chimeras of Cx50 and Cx46 which point to domains NT, TM1, E1, TM2 and CL as being responsible for open channel properties; pore lining. We also report single channel SCAM data for TM2 and TM3 indicating that these transmembrane domains are not pore lining.